The role of the dsRBP STAU1 in circRNA-mRNA interactions

Circular RNAs (circRNAs) are a class of long non-coding RNAs (lncRNAs) composed of single- stranded RNA molecules forming a covalently closed circular structure without free ends. CircRNAs can influence gene expression by interacting with DNA, RNA, and proteins and have been implicated in various pathological conditions, such as cancer. Our lab has explored the possibility of circRNA-mRNA interaction as a novel circRNA function in cancer cells. Recently, two functional circRNA-mRNA interactions have been described in rhabdomyosarcoma, the most common form of soft tissue sarcoma in childhood. CircHIPK3 interacts with BRCA1 mRNA, preventing the binding of FMRP1 protein, thus increasing its stability and translation. Conversely, circZNF609 interacts with CKAP5 mRNA and the HuR protein, facilitating HuR's loading onto CKAP5 mRNA and enhancing its stability. The aim of my PhD project is to characterize the role of proteins that potentially bind RNA-RNA duplexes produced by circRNA-mRNA interactions in RD cells, a cellular model of rhabdomyosarcoma. Double Stranded RNA Binding Proteins (DSRBPs) are a family of proteins known to bind dsRNAs through their Double Stranded RNA Binding Domains (DSRBDs). Among them, I am focusing on STAU1, a DSRBP involved in various aspects of post-transcriptional gene regulation. Firstly, I am employing a CLiPPR approach to discover new circRNA-mRNA interactions in RD cells. This experiment relies on the crosslinking of direct RNA-RNA duplexes using AMT, a Psoralen-derivative that allows to crosslink RNA molecules in close proximity. After crosslinking, an RNA pulldown with oligo dT is performed to specifically enrich polyadenylated RNA molecules. The RNA is then treated with or without RNAse R (an exonuclease that degrades linear RNAs but not circRNAs), followed by RNA-seq analysis. Comparing the RNAse R-treated sample with the untreated sample allows identification of putative mRNA interactors of circRNAs using BLAST or IntaRNA algorithms to align RNA sequences. In parallel, I am conducting a CLIP-seq experiment of STAU1. Following UV-C crosslinking of RD cells, an immunoprecipitation of STAU1 is performed to find the direct RNA interactors of the protein. As in the previous experiment, RNA samples are treated with RNAse R to enrich circRNAs for subsequent RNA-seq analysis. By merging the circRNAs found in the CLIPPR analysis and the STAU1 CLIP, the aim is to identify the circRNAs that directly interact with mRNAs and that are bound by STAU1 in RD cells. To understand the types of RNA-RNA duplexes recognized by STAU1, I have generated a series of vectors that mimic the RNA-RNA interactions of circHIPK3-BRCA1 mRNA and circZNF609- CKAP5 mRNA, as well as perfect RNA-RNA matches and negative controls. Through RIP experiments of STAU1, I aim to explore the types of molecules bound by STAU1 in RD cells (single-stranded RNA molecules, double-stranded RNA molecules with mismatches, and perfect double-stranded RNA molecules). As a future perspective, I plan to explore the role of other DSRBPs in circRNA-mRNA interaction dynamics, such as ADAR1, ADAR2, NFAR1, PKR, and others. In conclusion, this research aims to shed light on the complex interplay between circRNAs and mRNAs, focusing on the largely unexplored role of the DSRBP STAU1, with the hope of uncovering novel therapeutic targets and strategies for cancer treatment.